Ethanol production

ABSTRACT

The present invention relates to the production of ethanol as a product of bacterial fermentation. In particular this invention relates to a novel method of gene inactivation and gene expression based upon homologous recombination.

This application claims benefit of U.S. Application Ser. No. 60/247,017,filed Nov. 13, 2000, and U.K. Application No. 0024554.8, filed Oct. 6,2000, both incorporated herein by reference.

This invention relates to the production of ethanol as a product ofbacterial fermentation. In particular this invention relates to a novelmethod of gene inactivation and gene expression based upon homologousrecombination.

Many bacteria have the natural ability to metabolise simple sugars intoa mixture of acidic and neutral fermentation products via the process ofglycolysis. Glycolysis is the series of enzymatic steps whereby the sixcarbon glucose molecule is broken down, via multiple intermediates, intotwo molecules of the three carbon compound pyruvate. The glycolyticpathways of many bacteria produce pyruvate as a common intermediate.Subsequent metabolism of pyruvate results in a net production of NADHand ATP as well as waste products commonly known as fermentationproducts. Under aerobic conditions, approximately 95% of the pyruvateproduced from glycolysis is consumed in a number of short metabolicpathways which act to regenerate NAD⁺ via oxidative metabolism, whereNADH is typically oxidised by donating hydrogen equivalents via a seriesof steps to oxygen, thereby forming water, an obligate requirement forcontinued glycolysis and ATP production.

Under anaerobic conditions, most ATP is generated via glycolysis.Additional ATP can also be regenerated during the production of organicacids such as acetate. NAD⁺ is regenerated from NADH during thereduction of organic substrates such as pyruvate or acetyl CoA.Therefore, the fermentation products of glycolysis and pyruvatemetabolism include organic acids, such as lactate, formate and acetateas well as neutral products such as ethanol.

The majority of facultatively anaerobic bacteria do not produce highyields of ethanol either under aerobic or anaerobic conditions. Mostfacultative anaerobes metabolise pyruvate aerobically via pyruvatedehydrogenase (PDH) and the tricarboxylic acid cycle (TCA). Underanaerobic conditions, the main energy pathway for the metabolism ofpyruvate is via the pyruvate-formate-lyase (PFL) pathway to give formateand acetyl-CoA. Acetyl-CoA is then converted to acetate, viaphosphotransacetylase (PTA) and acetate kinase (AK) with theco-production of ATP, or reduced to ethanol via acetaldehydedehydrogenase (AcDH) and alcohol dehydrogenase (ADH). In order tomaintain a balance of reducing equivalents, excess NADH produced fromglycolysis is re-oxidised to NAD⁺ by lactate dehydrogenase (LDH) duringthe reduction of pyruvate to lactate. NADH can also be re-oxidised byAcDH and ADH during the reduction of acetyl-CoA to ethanol but this is aminor reaction in cells with a functional LDH. Theoretical yields ofethanol are therefore not achieved since most acetyl CoA is converted toacetate to regenerate ATP and excess NADH produced during glycolysis isoxidised by LDH.

Ethanologenic microorganisms, such as Zymomonas mobilis and yeast, arecapable of a second type of anaerobic fermentation commonly referred toas alcoholic fermentation in which pyruvate is metabolised toacetaldehyde and CO₂ by pyruvate decarboxylase (PDC). Acetaldehyde isthen reduced to ethanol by ADH regenerating NAD⁺. Alcoholic fermentationresults in the metabolism of 1 molecule of glucose to two molecules ofethanol and two molecules of CO₂. DNA which encodes both of theseenzymes in Z. mobilis has been isolated, cloned and expressedrecombinantly in hosts capable of producing high yields of ethanol viathe synthetic route described above. For example; U.S. Pat. No.5,000,000 and Ingram et al (1997) Biotechnology and Bioengineering 58,Nos. 2 and 3 have shown that the genes encoding both PDC (pdc) and ADH(adh) from Z. mobilis can be incorporated into a “pet” operon which canbe used to transform Escherichia coli strains resulting in theproduction of recombinant E. coli capable of co-expressing the Z.mobilis pdc and adh. This results in the production of a syntheticpathway re-directing E. coli central metabolism from pyruvate to ethanolduring growth under both aerobic and anaerobic conditions. Similarly,U.S. Pat. No. 5,554,520 discloses that pdc and adh from Z. mobilis canboth be integrated via the use of a pet operon to produce Gram negativerecombinant hosts, including Erwina, Klebsiella and Xanthomonas species,each of which expresses the heterologous genes of Z. mobilis resultingin high yield production of ethanol via a synthetic pathway frompyruvate to ethanol.

U.S. Pat. No. 5,482,846 discloses the simultaneous transformation ofmesophilic Gram positive Bacillus sp with heterologous genes whichencode both the PDC and ADH enzymes so that the transformed bacteriaproduce ethanol as a primary fermentation product. There is nosuggestion that bacteria transformed with the pdc gene alone willproduce ethanol.

EP-A-0761815 describes a method of homologous recombination whereby asporulation gene is inserted into Bacillus thurengiensis.

EP-A-0603416 describes a method of homologous recombination whereby anarbitary gene is inserted into Lactobacillus delbrueckii.

EP-A-0415297 describes a method of producing Bacillus strains expressinga mutant protease.

Biwas et al., (J. Bacteriol., 175, 3628-3635, 1993) describes a methodof homologous recombination whereby Lactococcus lactis has a chromosomalgene replaced by a plasmid carried modified copy. The method uses athermosensitive plasmid and cannot be used to transform a thermophilicbacterium.

A key improvement in the production of ethanol using biocatalysts can beachieved with thermophilic microorganisms that operate at hightemperature. The conversion rate of carbohydrates into ethanol is muchfaster. For example, ethanol productivity in a thermophilic Bacillus isup to ten-fold faster than a conventional yeast fermentation processwhich operates at 30° C. Consequently, a smaller production plant isrequired for a given volumetric productivity, thereby reducing plantconstruction costs. At high temperature, there is a reduced risk ofcontamination in the fermenter from other microorganisms, resulting inless downtime, increased plant productivity and a lower energyrequirement for feedstock sterilisation. Moreover, fermentation coolingis not required, reducing operating costs further. The heat offermentation helps to evaporate ethanol, which reduces the likelihood ofgrowth inhibition from high ethanol concentrations, a common problemwith most bacterial fermentations. Ethanol evaporation in the fermenterhead space also facilitates product recovery.

The inventors' strain originates from a wild-type isolate that is anatural composting organism and far more suited for the conversion ofsugars found in agricultural feedstocks to ethanol than traditionalmesophilic microorganisms. The base strain possesses all the geneticmachinery for the conversion of hexose and pentose sugars, andcellobiose to ethanol; the inventors have simply blocked the LDH pathwayto increase ethanol yields. This process is called self-cloning and doesnot involve expression of foreign DNA. Consequently, the resultingorganism does not fall under the safety regulations imposed on the useof genetically modified organisms (GMOs).

In contrast, conventional biocatalysts are either good ethanol producersunable to utilise pentose sugars or poor ethanol producers that canutilise pentose sugars. These organisms have been genetically modifiedusing complex genetic techniques so that they can convert both hexoseand pentose sugars to ethanol. However, there are doubts about thestability of these recombinant organisms and concerns over safety sincesuch organisms fall under the GMO safety regulations. Moreover,recombinant mesophiles have expensive nutrient requirements and aresensitive to high salt concentrations and feedstock inhibitors.

The metabolic reactions leading to lactic acid formation (LDH pathway)have been blocked by chemical mutagenesis and the resulting strain TN islactate negative and produces ethanol in high yield. However, the mutantstrain is unstable and spontaneously reverts to the lactacte-producingwild-type. Revertants grow faster than the mutant at low pH and in highsugar concentrations, and rapidly ‘take-over’ in continuous culture.During ‘take-over’, the main fermentation product changes from ethanolto lactate.

The inventors initiated a molecular biology program to tackle thestability problem and gain a better insight into the genetic systemsinvolved in ethanol formation. The inventors first developed genetictechniques to specifically manipulate the organism and a sporulationdeficient mutant amenable to genetic manipulation was then selected incontinuous culture. The inventors then sequenced several key metabolicgenes; lactate dehydrogenase (ldh), lactase permease (lp), alcoholdehydrogenase (adh) and a novel insertion sequence located within theldh gene. DNA sequence analysis of the ldh gene from the chemicallymutated strain revealed that the gene had been inactivated by theinsertion of a naturally occurring insertion sequence element (IE) (alsoreferred to as an IS element) in the coding region of the gene.Transposition into (and out of) the ldh gene and subsequent geneinactivation is itself unstable, resulting in reversion.

The inventors determined that the IE sequence within the ldh geneprovides a large area for homologous recombination. It was thereforeproposed that the stability of the ldh mutation could be improved byintegration of plasmid DNA into the IE sequence already present withinthe ldh gene of strain TN.

The stability of the ldh gene mutation was improved by specifichomologous recombination between a plasmid and the insertion sequencewithin the ldh gene. The resulting strain is a sporulation deficient,facultatively anaerobic, Gram-positive Bacillus which exhibits improvedethanol production characteristics in continuous culture. Results showthat this new type of mutant is completely stable and has superiorgrowth characteristics and ethanol productivity than the first mutantsgenerated by chemical mutagenesis.

Strain improvement has been achieved through a novel method of geneintegration based on homologous recombination. The site of integrationand plasmid for recombination can also be used to integrate andoverexpress native or heterologous genes.

Southern hybridisation studies indicated that 3 copies of a transposableinsertion sequence element (IE) are present on the chromosome ofBacillus strain LLD-R. The insertion sequence is 3.2 kb long andcomprises three DNA open reading frame sequences (ORF's) that arepotentially translatable into proteins. ORF1 exhibits no homology to anyprotein in the National Center for Biotechnology Information (NCBI)database (www.ncbi.nlm.nih.gov/) whereas istA and istB displaysignificant homology to a family of known transposase enzymes. Bacillusstrain TN was developed from LLD-R following chemical mutagenesis (FIGS.9A and 9B), and one copy of the insertion sequence was found within thestructural ldh gene resulting in inactivation of the ldh gene and alactate negative phenotype, the main metabolic product of fermentationthereby changing from lactate to ethanol. The DNA sequence of the ldhgene and the IE sequence (underlined) from Bacillus strain TN are shownin FIG. 1. The amino acid sequence of L-LDH is shown in FIG. 11.

However, this insertion proved to be relatively unstable and the mutantstrain TN spontaneously reverts back to strain TN-R with a functionalldh gene. The main metabolic product of fermentation changes fromethanol to lactate as shown in FIG. 2 which shows the geneticinstability of Bacillus mutant strain TN.

The IE sequence was amplified from TN chromosomal DNA by PCR. Primerswere chosen from the ldh gene sequence that flanked the insertionsequence and a HindIII restriction site was introduced into the upstreamprimer and a XbaI restriction site was introduced into the downstreamprimer to create convenient restriction sites for subsequent cloninginto plasmid pUBUC. A 3.2 kb PCR fragment containing the insertionsequence was trimmed using HindIII and XbaI restriction endonucleasesand subsequently cloned into plasmid pUBUC resulting in plasmid pUBUC-IE(FIG. 5).

In vivo methylation of plasmid DNA to prevent its restriction aftertransformation of Bacillus sp. was achieved after transformation,propagation in and purification from E. coli TOP10 cells harbouringplasmid pMETH. Methylated pUBUC-IE was then used to transform Bacillusstrain TN. Transformants were first isolated on TGP agar plates(kanamycin) at 52° C. Transformants were then screened using PCRamplification of the ldh gene. Failure to amplify a PCR product (greaterthan 10 kb using set PCR conditions) using LDH primers suggested that atleast one copy of the plasmid had become integrated into the chromosome.

The new strain, TN-T9, was grown under pH controlled conditions incontinuous culture without kanamycin selection to check for strainstability. Stability of strain TN-T9 was confirmed using sub-optimalfermentation conditions such that residual sugar was present within thefermentation medium; conditions which favour reversion. The fermentationran continuously for 750 hours without any trace of lactate productiondespite the presence of residual sugar within the fermentation medium,pyruvate excretion and numerous deviations from the set conditions.Ethanol was produced in relatively large amounts throughout thefermentation FIG. 4, indicating that the ldh gene mutation in strainTN-T9 is stable in continuous culture under the experimental conditionsprovided.

The inventors have also optimised the fermentation conditions for cellgrowth and ethanol production for Bacillus strain TN-T9.

In summary the inventors have developed a dual system for improving thestability of the ldh mutant whilst expressing pdc and adh genesoptionally using a pdc/adh operon. The inventors have also isolated andsequenced a novel ldh gene and insertion sequence element, as well asnovel lactate permease and alcohol dehydrogenase genes. Furthermore, theinventors have developed a technique for the integration of plasmid DNAinto the chromosome and selection of recombinant Bacillus sp and havedeveloped a set of optimised conditions for the production of ethanol bybacterial fermentation.

Accordingly, a first aspect of the present invention relates to arecombinant thermophilic, Gram-positive bacterium which has beentransformed using a method of homologous recombination for stabilising agene mutation and for inserting an expressible gene.

The invention also provides a recombinant thermophilic, Gram-positivebacterium in which the stability of the ldh mutation has been enhancedby homologous recombination between a plasmid and the chromosomal DNA ofthe bacterium resulting in a strain for the production of ethanol as aproduct of bacterial fermentation.

Preferably, the Gram-positive bacterium is a strain of B.thermoglucosidasius.

Preferably, the Gram-positive bacterium has been transformed with aplasmid harbouring an IE sequence as set forth in FIG. 1, or afunctional portion or variant thereof. Advantageously, the IE sequenceof FIG. 1, or functional variant or portion thereof, is stablyincorporated into the chromosome of the recombinant bacterium byhomologous recombination.

Preferably, integration of the IE sequence into the chromosome of therecombinant bacterium will result in the inactivation of the native ldhgene.

Preferably, the Gram-positive bacterium is Bacillus strain TN-T9 (NCIMBAccession no. NCIMB 41075 deposited on 8 Sep. 2000 in accordance withthe terms of the Budapest Treaty).

Alternatively, it is preferred that the Gram-positive bacterium isBacillus strain TN-TK (NCIMB Accession no. NCIMB 41115 deposited on 27Sep. 2001 in accordance with the terms of the Budapest Treaty).

The present invention also relates to a Gram-positive bacterium obtainedby selecting mutants of TN-T9 which are kanamycin sensitive. A suitablemethod for obtaining such strains is described in the appended examples.

Preferably, the Gram-positive bacterium is sporulation deficient.

According to a second aspect of the present invention there is provideda Gram-positive bacterium wherein a native ldh gene has been inactivatedby homologous recombination and one or more expressible genes have beeninserted into the chromosomal DNA of the bacterium. Furthermore, geneexpression may be increased by increased gene copy number followingmultiple insertions of the plasmid into the insertion sequence either asa result of one round or repeated rounds of integration.

The one or more expressible genes may be inserted into one or more IEsequences present in the chromosomal DNA of the bacterium. For example,there are 3 IE sequences on the chromosome of strains TN-T9 and TN-TK.

The gene to be expressed may be native to Bacillus such as alcoholdehydrogenase or foreign (i.e. heterlogous such as pyruvatedecarboxylase from Z. mobilis and α-amylase from B. stearothermophilus.The genes may also be arranged in an operon under the sametranscriptional control. Gene expression may be regulated bymanipulating the copy number of the gene and by using differenttranscriptional promoter sequences.

Preferably, the one or more genes are pdc and/or adh.

The amino acid sequence of adh is shown in FIG. 12.

According to a third aspect of the invention there is provided a methodof inactivating a native ldh gene and inserting one or more expressiblegenes into the chromosome of a bacterium by homologous recombination.

Preferably the bacterium is a thermophilic Gram-positive bacterium.

Preferably, the gene to be inactivated is a native ldh gene and the oneor more expressible genes are a pdc gene and a adh gene.

Preferably, the pdc gene and the adh gene form part of a PDC operonoperatively linked to the IE sequence of FIG. 1 on the same plasmid.

Preferably the pdc gene is heterologous to the cell.

Preferably, both the IE sequence of FIG. 1 and the PDC operon, orportions thereof, are stably integrated into the chromosome of thebacterium.

Advantageously, the method of gene inactivation and expression comprisesthe use of a shuttle vector, as set forth in FIG. 5, which is able toreplicate in E. coli and Bacillus strains at temperatures up to 54° C.

According to a fourth aspect of the present invention there is provideda shuttle vector which is able to replicate in both E. coli and Bacillussp at temperatures up to 54° C., which confers resistance to ampicillinand kanamycin and which harbours the IE sequence, or a portion thereofas set forth in FIG. 1, from Bacillus strain TN.

Preferably, the shuttle vector is pUBUC-IE as set forth in FIG. 5.

Preferably, the shuttle vector will contain a PDC operon comprising apdc gene and a adh gene under the control of the ldh promoter andoperably linked to the IE sequence of FIG. 1.

According to a fifth aspect of the present invention there is provided amethod of selecting for recombinant Bacillus sp at high temperaturewherein plasmid DNA has been stably integrated into the ldh gene of therecombinant bacterium by homologous recombination, comprising use of PCRto select for recombinants that do not contain the native ldh gene andIE sequence.

Preferably, successful integration of the insertion sequence into theldh gene will be indicated by failure to amplify a PCR product from theldh gene of the recombinant bacterium.

The present invention also provides one or more polypeptides encoded bythe sequence shown in FIG. 1 from nucleotide 652 to nucleotide 3800, ora functional variant or portion thereof, wherein the one or morepolypeptides have the biological activity of a transposase.

The one or more polypeptides may have the biological activity of atransposase taken alone or when combined with other polypeptides.

Preferably, the one or more polypeptides has the amino acid sequenceshown in FIG. 13, FIG. 14 or FIG. 15 or a functional portion or variantthereof.

The functional portions or variants retain at least part of thetransposase function of the polypeptide shown in FIG. 13, FIG. 14 orFIG. 15. Preferably the portions are at least 20, more preferably atleast 50 amino acids in length. Furthermore, it is preferred that thevariants have at least 80%, more preferably at least 90% and mostpreferably at least 95% sequence homology with the polypeptide shown inFIG. 13, FIG. 14 or FIG. 15. Homology is preferably measured using theBLAST program.

According to a sixth aspect of the invention there is provided a DNAsequence as set forth in FIG. 6, or a functional variant thereof, whichcodes for a polypeptide having the biological activity of the enzymelactate dehydrogenase.

According to a seventh aspect of the present invention there is provideda DNA sequence as set forth in FIG. 7B, or a functional variant thereof,which codes for a polypeptide having the biological activity of theenzyme lactate permease.

According to an eigth aspect of the present invention there is provideda DNA sequence as set forth in FIG. 8, or a functional variant thereof,which codes for a polypeptide having the biological activity of theenzyme alcohol dehydrogenase.

In this specification, functional variants include DNA sequences whichas a result of sequence additions, deletions or substitutions, or whichby virtue of the degeneracy of the genetic code, hybridise to and/orencode a polypeptide having a lactate dehydrogenase lactate permease oralcohol deydrongenase activity. Preferably, the variants have at least80%, more preferably 90% and most preferably 95% sequence homology tothe sequence shown in the Figures. Homology is preferably measured usingthe BLAST program.

A ninth aspect of the invention also provides a method for improving thestability of the ldh mutant comprising expressing genes using a pdc/adhoperon.

A tenth aspect of the present invention relates to a technique for theintegration and selection of recombinant Bacillus sp in accordance withthe invention.

According to the final eleventh aspect of the present invention there isprovided a process for the production of ethanol by bacterialfermentation of the Gram-positive bacterium of the present inventioncomprising optimised fermentation conditions of pH, temperature, redoxvalues and specific dilution rates for cell growth and ethanolproduction. Preferably, the fermentation conditions will comprise a pHrange of between 5.5-7.5 and a temperature range of 40-75° C. with redoxvalues being between −360-400 mV and dilution rates between 0.3 and 0.8h⁻¹.

BRIEF DESCRIPTION OF THE DRAWINGS

The production of recombinant bacteria in accordance with the presentinvention will now be described, by way of example only, with referenceto the drawings in which:

FIG. 1 shows the nucleotide sequence of a DNA sequence comprising aninsertion element (IE), wherein the IE sequence is underlined;

FIG. 2 is a schematic representation of the genetic instability ofstrain TN;

FIG. 3 is a schematic representation of the method for LDH geneinactivation by single-crossover recombination in Bacillus mutant strainTN;

FIG. 4 is a graphical representation showing the stability of Bacillusmutant strain TN-T9 in continuous culture for over 750 hours;

FIG. 5 is a schematic representation of shuttle vector pUBUC-IE;

FIG. 6 shows the DNA sequence of a novel lactate dehydrogenase gene andtranslated amino acid sequence from Bacillus strain LN;

FIG. 7A shows the partial DNA sequence of a novel lactate permease geneand the translated amino acid sequence from Bacillus strain LN;

FIG. 7B shows the full DNA sequence of a novel lactate permease gene andthe translated ammo acid sequence from Bacillus strain LN;

FIG. 8 shows the DNA sequence of a novel alcohol dehydrogenase gene(underlined) from Bacillus strain LN;

FIG. 9 is a schematic representation showing (A) the development ofBacillus strain TN-T9 and (B) the development of Bacillus strains TN-T9and TN-TK;

FIG. 10 shows the construction of an artificial PDC operon;

FIG. 11 shows the amino acid sequence of L-lactate dehydrogenase (ldh)from the TN strain;

FIG. 12 shows the amino acid sequence of alcohol dehydrogenase (adh)from the TN strain;

FIG. 13 shows the amino acid sequence of a transposase encoded by the IEsequence.

FIG. 14 shows the amino acid sequence of a transposase encoded by the IEsequence.

FIG. 15 shows the amino acid sequence of a transposase encoded by the IEsequence.

EXAMPLES

Materials and Methods

Construction of Plasmid pUBUC

A shuttle vector for the transfer of DNA between E. coli and theinventor's thermophilic Bacillus strains was developed by fusingplasmids pUC18 and pUB110. Plasmid pUB110 is a widely used vector thatwas isolated from Staphyloccocus aureus which confers resistance tokanamycin and which can replicate in B. stearothermophilus attemperatures up to 54° C. Narumi et al., 1992 Biotechnology Techniques6, No. 1. Plasmids pUB110 and pUC18 were linearised with EcoR1 andBamH1, and then ligated together to form pUBUC (6.4 kb). Plasmid pUBUChas a temperature sensitive replicon, and cannot replicate above 54° C.making it an ideal host for gene integration, via homologousrecombination at elevated temperatures.

Construction of Plasmid pMETH

A 1.1 kb fragment containing the met gene was amplified from Haemophilusaeygptius chromosomal DNA by PCR. The gene was verified by DNAsequencing. The met gene was trimmed with BamHI and XbaI, and thensubcloned into the expression plasmid pCL1920, previously linearisedwith BamH1 and XbaI. The resultant plasmid pMETH was transformed into E.coli TOP 10. E. coli TOP 10 cells harbouring pMETH were propagated andthe culture was harvested for subsequent transformation and in vivomethylation using a method described by Tang et al (1994) Nuc. Acid Res.22 (14). Competent cells were stored in convenient aliquots at −70° C.prior to transformation.

PCR Amplification

The IE sequence was amplified from TN chromosomal DNA by PCR usingprimers LDH7 and LDH8. The concentration of reactants and the PCRprocedure used were those recommended in the Expand™ High Fidelity PCRSystem (Roche Diagnostics). PCR amplification from lyophilised cells wasachieved after 30 cycles in a Genius thermocycler (Techne, Ltd.,Cambridge). The sequence of the upstream primer, LDH7, was 5′-AAGCTT GATGAA ATC COG ATT TGA TGG-3′ and the sequence of the downstream primer,LDH8 was 5′-TCTAGA GCT AAA TTT CCA AGT AGC-3′. These primers were chosenfrom the ldh gene sequence that flanked the insertion sequence. AHindIII restriction site was introduced into the upstream primer and aXbaI restriction site was introduced into the downstream primer tocreate convenient restriction sites for subsequent cloning (introducedsites are underlined).

Construction of Plasmid pUBUC-IE

The manipulation, transformation and isolation of plasmid DNA in E. coliwas performed using standard procedures (Maniatis). A 3.2 kb PCRfragment containing the insertion sequence was trimmed with HindIII andXbaI and then cloned into plasmid pUBUC. The resulting shuttle plasmid,referred to as pUBUC-IE (FIG. 5) can replicate in E. coli and Bacillusstrains at temperatures up to 54° C., confers resistance to ampicillinand kanamycin, and harbours the IE sequence from Bacillus strain TN.

Construction of PDC Operon

Bacillus strain TN converts the intracellular metabolite pyruvate toacetyl-CoA via the PFL or PDH pathway. Acetyl-CoA is then reduced toacetaldehyde and then to ethanol in reactions catalysed by AcDH and ADH,respectively. The introduction of a foreign PDC enzyme provides thecells with an alternative pathway for ethanol production that involvesdecarboxylation of pyruvate by PDC to form acetaldehyde which is thenreduced to ethanol by the native ADH enzyme. Both PDC and ADH areinvolved in the conversion of pyruvate to ethanol.

We have shown that expression of Z. mobilis pdc from plasmid pZP-1improves cell growth and stability of the mutant strain TN. However, wedid not see any significant increase in ethanol formation. Therefore, wedecided to increase pdc gene expression and co-express the native adhgene from Bacillus TN.

In plasmid pZP-1, the pdc gene was placed under the control of the ldhpromoter sequence from B. stearothermophilus NCA1503. We decided tochange the promoter with the ldh promoter from Bacillus LN (construct1). We then placed the adh gene from Bacillus strain LN under thecontrol of the ldh promoter (construct 2). Finally, both pdc (from Z.mobilis and adh (from Bacillus LN) were placed under the control of theldh promoter sequence (construct 3). All the genes have been amplifiedby PCR from Z. mobilis (pdc) and Bacillus strain LN (ldh promoter andpdc), trimmed with the appropriate restriction enzymes, ligated togetherand cloned into an E. coli plasmid vector. The 3 constructs were clonedinto the replicative shuttle vectors pUBUC, pFC1 or the integrativeshuttle vector pUBUC-IE for chromosomal integration.

Example 1

Transformation of TN

Plasmid pUBUC-IE was methylated in vivo after transformation,propagation in and purification from E. coli TOP10 cells harbouringplasmid pMETH. Methylated pUBUC-IE was then used to transform Bacillusstrain TN. Bacillus strain TN cells were grown at 65° C. in 50 ml of TGPmedium until the absorbance at 600 nm (A₆₀₀) reached 0.5-0.6. Theculture was chilled on ice for 15-30 min. The cells were harvested bycentrifugation and washed once in 10 ml and twice in 5 ml of cold THbuffer (272 mM trehalose and 8 mM HEPES; pH 7.5 with KOH). The cellpellet was re-suspended in 400 μl of TH buffer and stored at 4° C. priorto electroporation. Methylated plasmid DNA was used to transform strainTN by electroporation based on a method previously described by Narumiet al (1992) Biotechnology Techniques 6(1). The competent cells weredispensed into 90 μl aliquots and mixed with 2 μl of methylated plasmidDNA (250 ng/μl). The mixture was transferred to cold electroporationcuvettes (0.2 cm electrode gap) and incubated on ice for 5 minutes. Thesuspensions were then subjected to a 2.5 kV discharge from a 25 μFcapacitor and the pulse control was set at 201 ohms (time constant, τ=5ms) using a EquiBio Easyject electroporator. The cells were immediatelytransferred to 5 ml of pre-warmed TGP, incubated at 52° C. for 1 hr, andplated on TGP agar (10 μg/ml kanamycin). The plates were incubated for24-48 hours at 52° C.

Selection of Recombinants

The following method was used to select for chromosomal integration ofthe temperature sensitive plasmid pUBUC-IE by homologous recombination.

-   1. Transformants were grown in 5 ml of TGP (kanamycin) medium at    52° C. for 24 hours.-   2. 50 ml of fresh TGP (kanamycin) medium was inoculated with 1 ml    from O/N culture and incubated in a shaking water bath at 52° C.    until a OD₆₀₀ was reached ˜0.5.-   3. 15 ml of the above culture was centrifuged at 4100 rpm for 5 min    at 5° C. and the pellet was resuspended in 150 μl of TGP (10:g/ml    kanamycin) medium and spread on TGP (kanamycin) plates.-   4. The plates were incubated at 68° C. for 16 hours.-   5. The isolated colonies were picked and analysed for plasmid    integration into the insertion sequence site by PCR.    Screening of TN Integrants

TN integrants were isolated at 68° C. Failure to amplify a PCR productusing LDH primers in TN integrants indicated that at least one copy ofplasmid pUBUC-IE had become integrated into the chromosome. As a resultof integration the new strain TN-T9 was found to be more stable withregard to ldh reversion and “take over” than the parental strain TN.

Stability of Strain TN-T9

The fermentation was run under sub-optimal conditions such that residualsugar was present in the medium; conditions which favour reversion. Thefermentation ran continuously for over 750 hours without any trace oflactate production despite residual sugar, pyruvate excretion andnumerous deviations from the set conditions. Ethanol was produced inrelatively large amounts throughout the fermentation. Kanamycin was notused to select for integratnts throughout the entire fermentation. Theseresults indicate that the ldh gene mutation in TN-T9 is stable incontinuous culture under the experimental conditions (pH 7.0, 65° C.with a 2% sugar feed).

Ethanol Yields and Productivity

The fermentation conditions have been optimised for ethanol productionfrom glucose, xylose and glucose/xylose based feedstocks. Culture type:continuous Temperature: 65° C. pH: 6.8 Sugar concentration in feed:2-10% Sparge gas: air Redox: >−350 mV (controlled through air flow rate)Dilution rate: 0.36-0.6 h⁻¹

Under these conditions the ethanol yields obtained were between 0.4-0.5g/g sugar. Ethanol productivities, using a dilution rate of 0.5 h⁻¹,were approximately 4 and 8 g ethanol/litre/hour on 2 and 4% sugar feeds,respectively.

Example 2

Selection of the Kanamycin Sensitive Strain—TN-TK

Bacillus TN-TK is a kanamycin sensitive derivative of TN-T9. This strainis completely stable with regard to the ldh mutation and an ideal hostfor plasmid borne expression involving kanamycin as a selectable marker.

TN-T9 was first grown at 68° C. for 24 hours in 5 ml of TGP supplementedwith kanamycin (10 μg/ml). Approximately 100 ml of culture was spread ontwo TGP (Km) agar plates and incubated overnight at 68° C. Severalhundred colonies were obtained and 100 were transferred to fresh TGP(Km) plates using a sterile toothpick. After overnight growth at 68° C.,the colonies were transferred (by replica plating) to fresh TGP platesand TGP (Km) plates and grown overnight at 68 C.

Two kanamycin sensitive colonies were obtained on TGP but not on thecorresponding TGP (Km) plate. The ldh gene regions from these colonieswere amplified by PCR and found to be comparable in size to thedisrupted ldh gene from TN-T9 (parental strain). PCR was used todemonstrate that the strains had lost the gene conferring resistance tokanamycin. One derivative referred to as TN-TK was chosen for furthergrowth experiments. These experiments confirmed that the kanamycinsensitivity and ldh mutation were completely stable.

1-12. (canceled)
 13. A method of inactivating a native gene encoding lactate dehydrogenase and inserting one or more expressible genes comprising homologous recombination.
 14. The method according to claim 13 wherein the one or more expressible genes are a gene encoding pyruvate decarboxylase and a gene encoding alcohol dehydrogenase.
 15. The method according to claim 14 wherein the gene encoding pyruvate decarboxylase and the gene encoding alcohol dehydrogenase form part of a pyruvate decarboxylase (PDC) operon operatively linked to an insertion element (IE) sequence comprising nucleotides 651-3800 of SEQ ID NO:1.
 16. The method according to claim 13 in which the gene encoding pyruvate decarboxylase is from a Zymomonas sp.
 17. The method according to claim 16 in which the gene encoding pyruvate decarboxylase is from Zymomonas mobilis.
 18. The method according to claim 15 wherein the gene encoding alcohol dehydrogenase is from Bacillus strain LN.
 19. The method according to any one of claims 13 to 18 wherein an insertion sequence and a PDC operon, or portions thereof, are stably integrated into a chromosome of a recombinant bacterium.
 20. The method according to any one of claims 13 to 14 comprising using a shuttle vector which is able to replicate in E. coli and Bacillus strains. 21-46. (canceled)
 47. The method of claim 20 wherein the shuttle vector is pUBUC-IE. 